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CryoJane User Manual
Customer Service 1-866-737-2540
(In continental US only)
Customer Service 1-314-446-2989
(International)
Instrumedics grants the purchaser of the CryoJane system
the rights to use the patented CryoJane materials
Patent # 5,444,105
CRYOJANE TAPE-TRANSFER
STEP-BY-STEP INSTALLATION INSTRUCTIONS
2
Absolute Alcohol
The following items are required for installation:
Hair Dryer
Gauze
Gloves
01 Remove release paper
from ECU
02 Place ECU on top left
corner of cryostat
03 Using absolute alcohol and gauze
clean chamber wall
04 Heat left wall for
Mech/Flash Unit & Oil Bath
placement Also heat the
exposed adhesive on the
mounting plate and oil bath
05 Place Oil Bath towards the
rear of the left chamber wall
06 Route Oil Bath & Mech/Flash Unit
Cables underneath Mech
*07 Place Mech/Flash Unit in
Chamber.
Place Mech/Flash Unit low in
chamber near the front wall.
(Placement near the freeze
bar is ideal in most cryostats)
08 Route cables in door bar
09 Place door bar with cables
onto front chamber ledge.
If required attach Door Bar Support Strip
underneath door bar
3
10 Adhere Cable holders
along cryostat top outer wall
11 ECU Rear with Cable &
Power Cord Connectors
12 Connect Mech/Flash Unit, Oil Bath
Cables to ECU
& Power cord to the ECU and wall outlet
Turn the ECU on
13 Mech/Flash Unit Cable
connected to ECU
14 External-Installation
Complete
Cryo-Vac Shown
15 Internal-Installation Complete
Cryo-Vac Shown
*The standard CryoJane Mech/Flash Unit installation is shown in Fig. 07.
The Microm HM 560 cryostat requires an S-Plate Mech and separate mounting
plate for installation.
S-Plate Mech/Flash Unit for the Microm HM 560 Cryostat
Mounting Plate locks onto S-Plate Mech/Flash Unit
Slide Nest Entrance
The entire assembly is adhered to the left cryostat wall as shown in Fig. 07.
Note:
The S-Plate Mech/Flash Unit must have enough clearance from the front
wall of the cryostat for slides to enter the Slide Nest.
4
Table of Contents
Instrumedics Frozen Section Process Overview ……………
7
Freezing Process …………………………………………………………. 7
CryoJane Process ………………………………………………………… 7
CryoJane System Components ……………………………………..
8
CryoJane ECU …………………………………………………………….
9
CryoJane Mech/Flash Unit Components ……………………….
10
CryoJane Operating Procedure ……………………………………
11
Background ………………………………………………………………. 11
Preparation ……………………………………………………………….. 11
Use ……………………………………………………………………….. 12
Steps for a Single Section ………………………………………… 12
Steps for Multiple Sections ………………………………………. 18
Summary of the Instrumedics Frozen Sectioning Process .
19
Special Fixation … … … … … … … … … … … … … … … … … … … … … …
20
Aqueous Fixation …… ………………………………………………….. 20
“Anhydrous” Fixation …………………………………………………….. 21
CryoJane Maintenance ………………………………………………..
22
ECU ……………………………………………………………………… 22
Mech/Flash Unit………………………………………………………
5
22
Troubleshooting Guide … … … … … … … … … … … … … … …
23
Troubleshooting the Mech/Flash Unit and ECU ………………………… 23
CryoJane Process Troubleshooting ………………………………………. 25
Troubleshooting Finished Slides …………………………………………. 27
Oil Bath Accessories Kit Components …………………………..
29
Oil Bath Accessory Kit Operating Procedure ……………….
30
Background ……………………………………………………………… 30
Preparation ………………………………………………………………. 30
Use ………………………………………………………………………
Storing a Tissue Block …………………………………………..
Sectioning a Stored Tissue Block ……………………………….
Oil Bath Accessory Kit Troubleshooting Guide …………..
6
31
31
31
32
Instrumedics Frozen Section Process Overview
Freezing Process - Conventional Freezing vs. Snap Freezing
In the conventional freezing method, the tissue is typically frozen at about -30°C. During
this relatively slow freezing process, the ice crystals that form are usually large compared
to cellular dimensions. These crystals can cause considerable displacement and structural
damage to the tissue.
The Gentle Jane device is designed to snap-freeze the tissue in approximately 8 - 10
seconds. The freezing agent can be liquid nitrogen, LN2 chilled isopentane, dry ice or a
thermoelectric cooler (Peltier device). The temperature at which the tissue is frozen
should be - 60°C or colder.
NOTE: Gentle Jane Snap-Freezing methods are described in a separate SnapFreezing User Manual.
CryoJane Process
The CryoJane process is built around three special methodologies:
1. The capture of an undistorted, thin, snap-frozen section on a special cold adhesive
tape.
2. The lamination of the captured section onto a cold glass microscope slide coated with
an ultraviolet light curable pressure sensitive adhesive.
3. The curing of the adhesive on the slide with an 8 millisecond ultraviolet flash, and the
subsequent removal of the adhesive tape, leaving a still frozen section firmly adhered
to the microscope slide.
The CryoJane system is designed to permit the facile application of these methodologies,
and can be adapted to virtually all model cryostats.
7
CryoJane System Components
FIGURE 1. CryoJane System Components
1. Mechanism/Flash Unit. See CRYOJANE MECH/FLASH UNIT
COMPONENTS for details.
2. Electronics Control Unit (ECU). See CRYOJANE ECU for details.
3. ½ inch diameter Mech/Flash UnitCable (connected to the Mech)
4. Door Channel
5. 2-Shelf Unit
6. Hand Roller
7. Adhesive Coated Slides
8. Tape Windows
9. Knife Facet Wipers
10. AC Power Cord (not shown)
OPTIONAL EQUIPMENT:
11. Protective Oil Accessories Kit
(see Oil Bath Accessories Kit sections in this manual)
12. Cryo-Vac-Away System
(see Cryo-Vac-Away User Manual)
CryoJane ECU
8
1
5
2
4
3
6
7
Figure 3. CryoJane ECU - front and left side
Figure 2. CryoJane ECU - back
1. On/Off Switch
2. Pad LED
3. Power LED (or Bath LED if optional Oil Bath Accessory Kit is installed).
4. Flash/Pad Connector
5. Optional Oil Bath Connector
6. Fuses
7. AC Power Cord Connector
CryoJane Mech/Flash Unit Components
9
FIGURE 4. CryoJane Mech Components
1. Blue/Gray Pad
2. Stainless Steel Spring Clip
3. Black Knob
4. Flash Tray
5. Lid
CryoJane Operating Procedure
10
Background
In conventional sectioning the operator usually guides the cut section over the knife using
a brush or an anti-roll device. Even with a skillful, steady hand, this step can be difficult,
especially if a 2 to 4 micron thick section is desired.
To mount the section, a room-temperature slide is touched to it. When contact is made
between the warm slide and the frozen section, instantaneous melting of the section
occurs, converting the ice crystals back into water. Surface tension causes the melted
section to adhere to the slide. When the ice melts, water fills the spaces formerly
occupied by the ice crystals, rehydrating the nucleoplasm and cytoplasm, causing
erythrocytes to lyse and soluble substances to be displaced. Flow of the melted solutions
can distort and displace fine structure. Often the melted section is then dried, during
which step, surface tension forces further distort, displace and collapse tissue structure.
Pathologists and clinicians have been trained to recognize normal and abnormal structure
against this background of degraded fine structure.
In contrast, the CryoJane process is designed specifically to avoid melting and air drying
and its associated artifacts. It uses a cold adhesive tape to support and capture the section
during the cutting step. The unthawed section on the tape is then transferred to a special
cold adhesive-coated slide. The section is not melted in the mounting step. The adhesive
coating on the slide is then polymerized, and the tape is peeled away, leaving the section
permanently anchored to the slide while still frozen.
Preparation
•
•
•
•
A day’s supply of Adhesive Coated Slides and Tape Windows should be in the
cryostat, chilled and ready for use.
The cryostat temperature should be -25°C to -30°C (depending on the cryostat
model).
The Pad light (left LED) on the ECU should be green.
The right LED should be red if it is labeled Power and green if it is labeled Oil Bath.
Use
Once a tissue specimen has been frozen and mounted in the microtome chuck, follow the
procedure outlined below.
11
Steps for a Single Section:
1. If the Cryo-Vac-Away is installed,
position the collection nozzle at the
knife edge and step on the foot switch
to automatically remove debris during
the trimming sequence. If the CryoNozzle
Vac-Away is not installed, trim the
block to the desired depth to obtain a
full block face and remove the debris
from the knife. Set the micrometer for
the desired section thickness and cut
two or three sections to be sure a full
Figure 5. Block trimming and debris removal.
section will be cut.
2. Take a pre-cooled, pouched, Adhesive
Coated Slide, and while holding it
inside the cryostat chamber, remove
the glass slide from the pouch.
Figure 6. Adhesive coated slide in pouch.
.
3. Holding the Slide at the frosted end,
rapidly peel off the protective mylar
film which covers the adhesive and
discard it.
Figure 7. Peeling mylar from adhesive coated
slide.
12
4. Place the Coated Slide, adhesive-sideup, on the Blue/Gray Pad of the Mech
by slipping the frosted end of the slide
under the stainless steel Spring Clip.
Figure 8. Adhesive Coated Slide on Blue/Gray
Pad.
5. Inside the cryostat, pick up a pink
Tape and peel away the protective
film covering the adhesive window.
Discard the protective film.
Figure 9. Exposing the Tape window.
6. Hold the Tape up by the narrow tab
with the adhesive side towards the
block face. Position the Tape in front
of the trimmed block so that the
bottom edge of the exposed adhesive
is aligned with the bottom edge of the
block. Place the Tape on the block
face making sure that the bottom
wider tab of the Tape overhangs the
knife. NO PORTION OF THE
ADHESIVE WINDOW SHOULD
OVERHANG THE BLOCK FACE.
TO AVOID MELTING THE
SURFACE OF THE TISSUE DO
NOT TOUCH THE PORTION OF
THE TAPE THAT COVERS THE
BLOCK FACE.
Figure 10. Placing Tape on block face.
13
7. Using the cold Hand Roller, laminate
the Tape to the face of the block by
moving the Roller up and down one to
two times and then left to right one to
two times. Some pressure is required
for good adhesion. When finished,
place the Hand Roller back to its
original position inside the cryostat.
Figure 11. Laminating the Tape to the block face.
8. Turn the flywheel slowly and evenly
to cut a section. If a sharp knife is
used, the section captured on the Tape
will be flat, intact, uncompressed and
can be as thin as two microns. No
need for a brush or an anti-roll
device!
Figure 12. Cutting the section.
14
9. Pick up the Tape at either tab and
transfer it to the Slide on the
Blue/Gray Pad making sure to keep it
down inside the cryostat to avoid
melting the section.
Figure 13. Handling the cut section.
10. Position the Tape, section-side-down,
on the Adhesive Coated Slide. For
multiple sections refer to the Steps for
Multiple Sections.
Figure 14. Positioning the Tape on the Slide.
11. Use the cold Hand Roller to laminate
the Tape to the Slide. Apply even
pressure on the Roller and roll it over
the section under the Tape two or
three times
Figure 15. Laminating the section to the Slide.
15
12. Holding the frosted end of the Slide,
remove it from the Blue/ Gray Pad
and transfer it into the Flash Tray.
Gently push the Slide in as far as it
will go, against the stop. (To check,
lift the Lid.) Close before actuating
the flash.
Figure 16. Placing the slide into the Flash Tray.
13. Push and release the Black Knob to
actuate the UV flash. The UV cures
the adhesive on the Slide into a
polymer, anchoring the frozen section
to the Slide.
DO NOT FLASH MORE THAT
ONCE EVERY 30 SECONDS.
MORE OFTEN CAN SERIOUSLY
DAMAGE THE ECU.
Figure 17. Curing the adhesive on the Slide.
14. Holding the frosted end, immediately
remove the cured Slide from the Flash
Tray. DO NOT TAKE THE SLIDE
OUT OF THE CRYOSTAT AT THIS
TIME AS THAT WILL CAUSE THE
SECTION TO MELT.
Figure 18. Removing the cured Slide.
16
15. Keeping the Slide deep inside the
cryostat chamber, carefully remove
the pink Tape to expose the
transferred frozen section on the Slide.
Figure 19. Removing Tape from cured Slide.
16. Peel off the Tape diagonally and
downward to minimize sheer stress on
the section and to insure complete
transfer. Do not touch the section
with your fingers. Once removed,
discard the Tape.
The slide can now be:
a) fixed in the Instrumedics Aqueous
Fixative,
b) freeze-dried or freeze-substituted in
the cryostat before “anhydrous”
fixation,
c) melted or air-dried and fixed with
the fixative of choice for your
protocol
Figure 20. Technique for removing Tape from
Slide.
Note:
The section should be
immersed in the room temperature
fixative inside the cryostat. Fixation
should then continue at room
temperature.
17
Steps for Multiple Sections:
17. Multiple sections can be positioned on
one Slide by placing the Tapes
perpendicular to the Slide. For small
tissue specimens, the Tape can be cut
into narrower strips. Follow Steps 5 - 9.
18. To cure the adhesive on the slide, lift
the Lid of the Mech and seat the Slide
on the Flash Tray. The Tape tabs will
overhang the Flash Tray.
Figure 21. Processing multiple sections.
19. Close the Lid and push and release the
Black Knob to actuate the UV flash.
Then lift the Lid, remove the Slide and
carefully peel away each strip of Tape
as described in Steps 15 and 16.
Figure 22. Curing multiple sections.
18
Summary of the Instrumedics Frozen Sectioning Process
Snap-freezing and embedding.
A blockholder is placed on the Gentle Jane snap-freezing device.
CryoGel or other embedding media is dispensed onto the blockholder
and a tissue specimen is positioned on top. The chilled heat extractor is
placed in its holder and released. When the heat extractor contacts the
specimen, the tissue and embedding medium is snap-frozen into a flat
block. The plane of the flat block face minimizes trimming. When
specimen shape or orientation are essential, the tissue is snap-frozen
using the Instrumedics CryoGel / Rubber Mold method.
FIGURE 23. To minimize ice-crystal
size, tissue is snap-frozen with a chilled
heat extractor.
Cutting
After the block is trimmed, a cold adhesive tape is adhered to the block
face. The tape supports and captures the section as it is being cut,
eliminating the need for a brush or anti-roll device.
FIGURE 24. A cold adhesive tape is
used to support the section as it is being
cut.
Transfer to Slide
A cold adhesive-coated slide is placed on a temperature-controlled pad.
The adhesive tape is placed section-side-down on the adhesive-coated
slide, and is laminated to the adhesive layer using a cold roller.
FIGURE 25. The still frozen section,
adhered to the tape is rolled onto the
cold adhesive-coated slide.
Curing the Adhesive Coating
FIGURE 26.
An ultraviolet flash
converts the adhesive coating into a hard
solvent-resistant plastic.
A flash of ultraviolet light passes through the slide to polymerize the
adhesive layer on the slide into a hard, solvent-resistant plastic, tightly
anchoring the section. to the slide.
Removal of Tape
FIGURE 27. The tape is removed.
Several options are available
The tape is peeled away leaving the still frozen section tightly bonded to
the plastic layer.
The slide can then be:
a) fixed in the Instrumedics Aqueous Fixative,
b) freeze-dried or freeze-substituted in the cryostat before “anhydrous”
fixation,
c) melted or air-dried and fixed with the fixative of your choice.
19
Special Fixation Protocols
Aqueous Fixative: THIS FIXATION SHOULD BE USED FOR ALL
HISTOLOGICAL STAINING OF CRYOJANE PREPARED SECTIONS.
This fixative melts and fixes the tissue simultaneously (direct controlled “melting”). Ice
crystal artifacts (holes) are masked. Nuclear morphology is usually excellent. Some cell
components are lost and most of the red blood cells are lysed.
1. PREPARATION OF AQUEOUS BUFFER
Dissolve Buffer-Salt Mix in 180 ml of water.
Buffer-Salt Mix is available from Instrumedics (Cat. #AB)
Store in refrigerator. Usable for at least 6 months.
2. PREPARATION OF AQUEOUS FIXATIVE
Add 20 ml of 25% glutaraldehyde to 30 ml of Aqueous Buffer.
Mix well.
Use at room temperature.
Discard after 1 week. (If using 50% glut. add 10 ml glut. and 10 ml water.)
CAUTION! Contains Cacodylic Acid. Avoid contact with fingers or skin.
Do not breathe dust from vial.
3. PROCEDURE
a) After the adhesive coating on slide has been polymerized, carefully peel off
the pink Tape.
b) To avoid uncontrolled melting and/or drying of the section, immediately bring
the room temperature aqueous fixative into the cryostat and immerse the slide
into the fixative. Dip 2-3 times.
c) Continue to fix for 15 to 30 seconds at room temperature.
d) Rinse the slide in water and proceed with a chosen staining protocol.
20
“Anhydrous” Fixation Protocol - To be used after “freeze-substitution” in a cold solvent
such as acetone at -30°C or colder.
“Anhydrous” fixation preserves most cellular components and is highly recommended for
routine stains. Note: Not recommended when ice crystal size is large and widespread
(poor freezing). In such cases the aqueous fixative protocol should be followed. An
“anhydrous” fixative contains a maximum of 30% water.
1. AN/DMF/Glut
Dimethylformamide (DMF)
Acetonitrile (AN)
glutaraldehyde
Add H2O to make up to
Wait at least 1 our before use.
2. Formal-Ethanol
Ethanol (100% alc.)
Formaldehyde
35 ml
35 ml (can use 70 ml DMF and no AN)
10 ml 25% Glut (5 ml 50% Glut)
100 ml
86 ml
14 ml
Bring the fixative into the cryostat and transfer the freeze-substituted slide directly from
the acetone bath into the fixative. Continue fixation at room temperature for 15 to 30
seconds. (Transfer is made within the cryostat to prevent condensation of water on the
cold slide prior to fixation.)
3. For very sensitive antigens: Fix in the cold for 1 min.
DMF/Glut
Dimethylformamide
70 ml
25% Glutaraldehyde
1 ml
100 ml
Add H2O to make up to
Transfer the freeze-substituted slide directly from acetone to the fixative at
-4 C for 30 to 60 seconds. This transfer must be made within the cryostat.
Following fixation, the slides are rinsed in water prior to IHC protocols
REFER TO REAGENT MSDS FOR SAFE HANDLING
21
CryoJane Maintenance
ECU
• NOTHING SHOULD BE PLACED OR STORED ON TOP OF THE ECU! Any
spills which enter the ECU may severely damage the electronic components.
• To clean the ECU, wipe the surfaces with either a dry rag, or if necessary, an alcohol
wipe. ACETONE OR OTHER SOLVENTS WILL DAMAGE THE PLASTIC
SURFACES.
• Fuses can be replaced by unscrewing the two small fuse holders in the rear of the
ECU. Fuses should be 0.75 AMP SLO-BLO , (available from Instrumedics).
DO NOT OPEN THE ECU! Only personnel authorized by Instrumedics should
service the ECU. Unauthorized opening of the ECU can cause serious injury and will
void all warranties. Unauthorized persons will assume all risks and responsibilities.
Mech/Flash Unit
• All metal surfaces may be wiped with alcohol or chlorine solution.
• The Flash Tray should periodically be cleaned with alcohol to remove any adhesive
build-up.
• Care must be taken to avoid damaging the glass filter in the Flash Tray.
• Liquid spills on the Mech/FlashUnit should be wiped immediately. Electronic
components in the Flash Lamp may short circuit if exposed to liquids.
• DO NOT IMMERSE THE MECH/FLASH UNIT IN ANY SOLUTIONS.
• To remove the Mech/Flash Unit from the wall for decontamination or servicing,
simply unscrew the two screws shown below and then remove the Mech from the
cryostat. The Mech/FlashUnit’s mounting plate will remain attached to the wall of
the cryostat. (Tip: To avoid losing the two screws, screw them into the holes in the
mounting plate remaining on the wall, attach it to the mounting plate with the two
screws.
.
22
Troubleshooting the Mech and ECU
♦ The LEDs (lights) on the ECU are not on.
Cause
Solution
Make sure the ECU is properly connected to an
electrical source. Make sure the On/Off Switch
The ECU is not turned on.
on the ECU is in the On position (pushed in).
If the LEDs are on that indicates that the ECU
has power.
Check the fuses on the back of the ECU and
One or both fuses in the back of the ECU are replace as needed. Use only 0.75 AMP, SLOblown.
BLO fuses (available from Instrumedics).
Call Instrumedics’ customer service number
The ECU is malfunctioning.
and arrange for servicing of the ECU. DO NOT
OPEN THE ECU!
♦ The left LED (labeled Pad) always displays a solid red light.
Cause
Solution
The ½ inch diameter Flash/Pad Power Cable is Make sure the Flash/Pad Power Cable is
not connected properly.
properly connected to the ECU and to the Blue
Pad connector underneath the Mech.
The temperature of the Blue/Gray Pad is too Raise the cryostat temperature.
cold.
Call Instrumedics’ customer service number
The ECU is malfunctioning.
and arrange for servicing of the ECU. DO NOT
OPEN THE ECU!
♦ The left LED (labeled Pad) always displays a flashing red light.
Cause
Solution
The temperature of the Blue/Gray Pad is too Lower the cryostat temperature.
warm.
If the sections are not melting on the pad ignore
the flashing red light
Call Instrumedics’ customer service. I
DO NOT REMOVE THE LID OF ECU!
THE ECU LID SHOULD ONLY BE
The ECU is malfunctioning.
23
REMOVED BY AUTHORIZED PERSONS
ONLY IF THE OIL BATH OPTION IS INSTALLED:
♦ The right LED (labeled Oil Bath) is always red.
Refer to the Oil Bath Accessory Kit Troubleshooting Guide in this manual.
♦ The Flash does not go on when triggered.
Cause
Solution
Make sure the ECU is properly connected to an
electrical source. Make sure the On/Off Switch
The ECU is not turned on.
on the ECU is in the On position (pushed in). If
the LED’s on the front of the ECU are on, that
indicates that the ECU is on.
The ½ inch diameter Flash/Pad Power Cable is Make sure the Flash/Pad Power Cable is
not connected properly.
properly connected to the matching connector
on the rear of the ECU.
Check the fuses on the back of the ECU and
The fuses may be blown
replace as needed. NOTE: If flash unit is
triggered more than 2 times in one minute, one
One or both fuses in the back of the ECU are or both fuses on the back of the ECU will blow
out and will need to be replaced. This is a
blown.
protective feature designed to prevent
overheating. Use only 0.75 AMP, SLO-BLO,
Ceramic Core Wound fuses (available from
Instrumedics).
Check for ice build-up around the Black Knob
on the Mech and remove if found. If this
The Flash triggering mechanism is jammed.
problem cannot be corrected, call Instrumedics’
customer service number and arrange for
servicing of the triggering
Call Instrumedics’ customer service number and
The flash unit is malfunctioning.
arrange for servicing of the flash unit.. DO
NOT OPEN THE FLASH UNIT!
Call Instrumedics’ customer service number and
The ECU is malfunctioning.
arrange for servicing of the ECU.
DO NOT OPEN THE ECU
24
CryoJane Process Troubleshooting
The CryoJane process will not produce the desired results if the cryostat and/or the
microtome are malfunctioning. Both need to be serviced periodically. Check with the
manufacturer/dealer.
♦ The Tape does not adhere to the block face.
Cause
Solution
The protective film covering the adhesive is not Peel away the protective film covering the
removed.
adhesive window which says “PEEL THIS
AWAY”
Check the temperature of the cryostat and
The block is too cold or too warm.
adjust if necessary. Chamber temperature
should generally be set between -25°C to -27°C
depending on the particular cryostat model.
There is moisture on the adhesive layer of the Check to see if the cryostat needs to be
tape or on the blockface
defrosted. In warm, humid weather keep the
The tape will not adhere to the blockface if cryostat lid closed as much as possible
there is condensation on either the tape or the The tape will not adhere to the blockface if
blockface.
there is condensation on either the tape or the
blockface.
♦ The Tape does not capture the section.
Cause
Solution
The microtome is not advancing.
The microtome is not advancing at the desired
thickness.
The knife angle is too small or too large.
Check the microtome and adjust.
Check the microtome and adjust. Cut two or
three sections before applying the Tape.
Adjust the knife angle between 3 and 5
degrees.
The Tape is defective.
Use another Tape.
The Tape does not stay adhered when the The Tape is overhanging the blockface and
section is being cut.
snagging on the knife which causes the tape to
pull away. Cut the tape so that it fits the block.
The blockface is not flat.
The block should be faced off (trimmed) so the
the blockface is flat. The Tape should be
adhered to the entire surface.
♦ The Tape is cut during sectioning.
Cause
Solution
The block face is not flat.
The adhesive on the Tape overhangs the block
and snags on the knife.
Cut the Tape so that is fits the blockface.
The knife and/or the block are not be properly
Align and trim the block to obtain a flat face.
Position the Tape on the block so that no
adhesive is exposed to the knife when cutting
the section.
Check the knife and block for looseness and
25
tightened.
tighten all screws and clamps as needed.
♦The captured section on the Tape is not intact.
Cause
Solution
The most common cause is the section is not Use pressure when you roll over the section to
laminated with enough pressure to get good laminate to the adhesive layer on the slide. If
adherence to the adhesive layer.
you are using CJ-1X slides you may want to try
CJ-4X
If using a disposable blade, shift to a different
The knife is dull.
portion of the blade’s edge or replace the blade.
If using a stainless steel or tungsten-carbide
knife, sharpen the knife.
There is dirt on the Tape
Use a fresh, clean and cold Tape.
Bubbles form while laminating the Tape to the Apply the Tape “like wallpaper” to the block
block face.
face while laminating with the cold Roller. If
bubbles are still visible, reapply the Tape.
There is vibration in the microtome (thick/thin, Check the knife and block for looseness and
chatter, etc.)
tighten all screws and clamps as needed.
♦The Transferred Section is not Intact. Parts of the Section Remain on the Tape.
Cause
Solution
The section does not transfer intact
Remove the Tape slowly and carefully, peeling
it diagonally and downward to minimize the
tension on the section.
The knife is dull.
If using a disposable blade, shift to a different
portion of the blade’s edge or replace the blade.
If using a stainless steel or tungsten knife,
sharpen the knife.
The Tape was not well adhered to the adhesive Pressure must be exerted when rolling the tape
layer on the slide in the laminating step.
with the section on to the adhesive layer on the
slide to insure the section is well adhered. This
is critical for good transfer.
A bubble may have formed when the Tape was Apply the Tape “like wallpaper” to the Slide
laminated to the Slide.
while laminating with the cold Roller.
26
Troubleshooting Finished Slides
♦ The polymerized adhesive picks up excessive red stain after H&E.
Cause
Solution
Eosin may be too acidic or too alcoholic. This Stain a shorter time, or use an aqueous eosin Y
may cause background staining.
at pH 5.0 - 5.2.
♦ There are chatter marks (“venetian-blind” effect, parallel to knife edge)
in the section.
Cause
Solution
There is looseness in the system
Check tightness of chuck. Clamp knife holder
and/or blade holder very tightly.
If using a disposable blade, shift to a different
portion of the blade’s edge or replace the blade.
If using a stainless steel or tungsten knife,
sharpen the knife.
Cut slowly without stopping. If motor drive is
used, set cutting speed slower.
The knife is dull.
Stop/start motion during cutting.
♦ There are tears in the section.
Cause
Solution
If using a disposable blade, shift to a different
The knife has a nick or defect on the cutting portion of the blade’s edge or replace the blade.
edge.
If using a stainless steel or tungsten knife,
sharpen the knife.
♦ Small clusters of cells (usually circular) are missing in the section.
Cause
Solution
Bubbles or debris was captured between the
adhesive Tape and the block face, or between
the Tape and the coated Slide.
A hole in the block face may be due to groups
of cells being “plucked-out” during or prior to
sectioning.
Debris was caught between the Tape and block,
or between the Tape and the coated Slide.
To avoid bubbles, apply Tape “like wallpaper”.
If bubbles are visible under the Tape on the
block face, reapply the Tape. Use clean Tape.
This may be a defect in the tissue block.
Check the knife edge for dullness and correct.
Always remove all trimming debris from the
block face, and both the front and rear surfaces
of the knife’s cutting edge before sectioning.
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♦ There are holes in the nuclei and/or cytoplasm of the section.
Cause
Solution
The freezing process is slow and the Snap-freeze tissue at -60°C or colder. Using
temperature in not low enough to prevent Instrumedics’ Stand Alone Gentle Jane snapdamaging large ice crystals from forming.
freezer with liquid nitrogen produces the best
results. Snap-freeze the thinnest practical
Section may have melted and refrozen causing specimen. The sections uppermost in the block
large ice crystals to grow. (This is the most will have the minimum ice crystal artifact.
common problem.)
Transfer the frozen block from the freezing
device rapidly to the cryostat to avoid thawing
the tissue.
Avoid finger contact with the central portion of
the Tape and/or Slide. Do not touch the block
face with your fingers (thumb). Do not breath
directly on the section. Keep Tapes and Slides
inside the cryostat.
The Blue/Gray Pad temperature may be too
warm - See the Mech and ECU
Troubleshooting Guide.
ALL STEPS OF THE CryoJane PROCESS
MUST BE CARRIED OUT DEEP INSIDE
THE CRYOSTAT CHAMBER TO AVOID
WARM AIR CONTACTING AND MELTING
THE FROZEN SECTION.
Use Instrumedics’ Aqueous Fixative.
NOTE: Excellent results with freezesubstitution followed by “anhydrous” fixation
will occur only if the ice crystal size is
minimal.
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Oil Bath Accessories Kit
FIGURE 28. Oil Bath Accessory Kit Components
1. Oil Bath
2. Protective Oil®
3. Oil Brush
4. Oil Bath Power Cable (not shown)
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Oil Bath Accessory Kit Operating Procedure
Background
Virtually all embedding media are water based. When a frozen tissue specimen
embedded in such media is stored, over time the tissue and the medium will dehydrate
and degrade. The Oil Bath Accessory Kit enables the user to coat the block face with the
Protective Oil that is maintained at -8°C in a bath inside the cryostat chamber. Coating
the surface will not melt the tissue in the block. The Protective Oil has a freezing
temperature of -10°C and will instantly freeze on the block face. The frozen Oil coating
prevents tissue and medium dehydration for up to one year if stored in a -70°C freezer.
The Protective Oil can also be used to repair a damaged block. A deformation or hole in
the block face may be filled with the Oil, which will freeze. The frozen Oil has the
property of an embedding medium and can then be sectioned similarly to other media.
Preparation
•
•
•
•
•
The cryostat temperature should be -25°C to -30°C.
Make sure there is Oil in the Bath. If Oil is added to the Bath, wait 1 to 2 hours for
the Oil to reach its set temperature of -8°C.
The Protective Oil inside the Oil Bath should be in a liquid state (not frozen).
The Oil Brush should be inside the Oil Bath.
The right LED on the ECU (labeled Oil Bath) should be green.
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Use
A tissue block can be stored in a freezer at -80°C for up to 1 year once thoroughly coated
with the Protective Oil.
THE BLOCK MUST NOT BE PERMITTED TO THAW AND REFREEZE DURING
STORAGE. THIS WILL DEGRADE THE QUALITY OF THE TISSUE IN THE
BLOCK
Storing a Tissue Block
To store (short term in the cryostat or long term in a freezer) a frozen tissue block follow
the procedure outlined below.
For Short Term Storage in the Cryostat
With the block in the cryostat, use the Oil
Brush to coat the block face with the Oil
from the Oil Bath.
For Long Term Storage in a -80°C
Freezer
With the block in the cryostat, use the Oil
Brush to coat the entire block with the
Protective Oil from the Oil Bath. Apply
generous amounts of Oil.
Figure 29. Coating the block with Oil.
Sectioning a Stored Tissue Block
If the block has been stored in a freezer at –80C place the block in the cryostat for at
least an hour before sectioning.
Trim away the frozen layer of Oil to expose the tissue specimen and proceed as usual.
31
Oil Bath Accessory Kit Troubleshooting Guide
♦ The Oil inside the Oil Bath is frozen.
Cause
Solution
Make sure the ECU is properly connected
to an electrical source. Make sure the
The ECU is not turned on.
On/Off Switch on the ECU is in the On
position (pushed in). If the LED’s on the
front of the ECU are on, that indicates that
the ECU is on.
The Oil Bath Power Cord is not connected Make sure the Oil Bath Power Cord is
properly.
properly connected to the ECU and the Oil
Bath.
One or both fuses on the back of the ECU Check the fuses on the back of the ECU
are blown.
and replace as needed. Use only 0.75 AMP,
SLO-BLO fuses (available from Instrumedics).
The cryostat temperature is too cold.
Raise the cryostat temperature.
Call Instrumedics customer service number
and arrange for servicing of the ECU DO
NOT OPEN THE ECU!
The ECU is malfunctioning.
♦ The Oil Bath LED always displays a solid red light.
Cause
Solution
The Oil Bath Power Cord is not connected Make sure the Oil Bath Power Cord is
properly.
properly connected to the ECU and the Oil
Bath.
The temperature of the Oil inside the Oil Raise the cryostat temperature.
Bath is too cold.
Call Instrumedics’ customer service and
The ECU is malfunctioning.
arrange for servicing DO NOT OPEN
THE ECU!
♦ The Oil Bath LED always displays a flashing red light.
32
Cause
Solution
The temperature of the Oil inside the Oil Lower the cryostat temperature.
bath is too warm.
Call Instrumedics customer service and
The ECU is malfunctioning.
arrange for servicing of the ECU. DO
NOT OPEN THE ECU!
33